Resin acids and Rosin acids, barium salts

Endpoint summary

Description of key information

The substance was a skin sensitizer in the LLNA (OECD 429, GLP). The EC3 was 12.8 (BASF 2020).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Sep. 2019 - Jan. 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

22 July 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS B.V., Kreuzelweg 53, 5961 NM Horst, The Netherlands
- Females (if applicable) nulliparous and non-pregnant: not specified
- Microbiological status of animals, when known: specific pathogen free (SPF)
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 16.1 g – 20.4 g (pretest), 16.8 g – 21.3 g (main test)
- Housing: single
- Diet (e.g. ad libitum): Mouse and rat maintenance diet “GLP”, Granovit AG, Kaiseraugst, Switzerland; ad libitum
- Water (e.g. ad libitum): Tap water; ad libitum
- Acclimation period: at least 5 days before the first application
- Indication of any skin lesions: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 – 24°C
- Humidity (%): 45 – 65%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 h / 12 h
- IN-LIFE DATES: From: 08. Oct. 2019 To: 14. Oct. 2019

Vehicle:

dimethylformamide

Concentration:

10%, 25% and 50% in pretest
5%, 10% and 25% (w/w) in the main study

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: highest technically used test-substance concentration was a 50% test-substance preparation.
- Irritation: no relevant signs after application of the 10% and 25% concentration; excessive ear skin irritation after application of the 50% concentration
- Systemic toxicity: no relevant signs after application of the 10% and 25% concentration; reduced general condition after application of the 50% concentration on study day 2
- Ear thickness measurements: no relevant signs after application of the 10% and 25% concentration; increased in ear weights (compared to historical vehicle values) and ear thickness measurements after application of the 50% concentration
- Erythema scores: no data

MAIN STUDY
- four test groups
- Randomization: Prior to first application, the animals were distributed to the individual groups (Win Rando Version 3.2).
- Body weight determination: Individual body weights on day 0 prior to the first application and on day 5 prior to the sacrifice of the animals.
- Signs and symptoms: Obvious signs of systemic toxicity and/or local inflammation at the application sites were noted for each animal in the raw data.
- Mortality: A check for moribund and dead animals was made twice each workday (beginning and end) and once on Saturdays, Sundays and on public holidays.
- Form of application: Epicutaneous
- Application volume: 25 μL per ear
- Site of application: Dorsal surface of both ears
- Frequency of application: 3 consecutive applications (day 0 – day 2) to the same application site
- ³H-thymidine injection: On study day five (about 66 to 72 hours after the last application of test substance to the ears), about 20 μCi 3H-thymidine in 250 μL sterile saline were injected into the tail vein of the mice.
- Terminal procedures
- The animals were sacrificed on study day 5 about 5 hours after 3H-thymidine injection by cervical dislocation under Isoflurane anesthesia.
- Determination of ear weight: Immediately after the death, a circular piece of tissue (diameter 0.8 cm) was punched out of the apical part of each ear of all animals. The weight of the pooled punches was determined for each animal. These measurements serve for detecting a potential inflammatory ear swelling.
- Removal and weight determination of the lymph nodes: Dissection of the left and right auricular lymph nodes. The weight of the pooled lymph nodes from both sides was determined for each animal.
- Preparation of cell suspension and determination of cell count: After weight determination, the pooled lymph nodes of each animal were stored in phosphate-buffered saline (PBS) in an ice water bath until further preparation. A single cell suspension was prepared as soon as possible after dissection by carefully passing the lymph nodes through an iron mesh (mesh size 200 μm) into 8 mL phosphate-buffered physiological saline. For determination of cell counts, an aliquot of each suspension was further diluted with Casy®ton in a ratio 1:500. The cell count was determined using a Casy® Counter.
- Measurement of 3H-thymidine incorporation of the lymph node cells: The remaining cell suspensions were washed twice with PBS and precipitated with 5% trichloro-acetic acid (TCA). Each precipitate was transferred to scintillation fluid and incorporation of 3H-thymidine into the cells was measured in a β-scintillation counter.

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: In order to reveal a possible induction of sensitization, the response in the draining lymph node after epicutaneous application of several concentrations of the test substance to the skin of the dorsal surface of both ears is determined by measuring 3H-thymidine incorporation into the lymph node cells. Additional parameters used to characterize the response are lymph node cell count and to a certain extent lymph node weight. As irritation by the test substance may also induce lymph node responses, the weights of ear punches taken from the area of test substance application are determined as an indicator for inflammatory ear swelling due to any irritant action of the test substance.
A test item is regarded as sensitizer in the LLNA if exposure to at least one concentration of the test item results in an incorporation of 3H-thymidine at least 3-fold or greater than that determined in control mice as indicated by the stimulation index (SI ≥ 3.0). However, the biological relevance of the obtained stimulation indices is considered in conjunction with the other assessed endpoints (i.e. lymph node cell counts, lymph node weights, ear weights).
Hereby, the thresholds used for assessment of cell counts and ear weights are represented by stimulation indices (SI) of ≥ 1.5 and ≥ 1.25, respectively. If applicable, the EC (estimated concentration) leading to the respective SI values were calculated by linear or semi-logarithmical regression between the data points directly below and above the SI if possible or by using the two nearest points below or above the SI.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Mean values and standard deviations: for measured parameters per test group
WILCOXON - Test: 3H-thymidine incorporation, cell count, lymph node weight and ear weight

Positive control results:

Studies using the positive control substance Alpha-Hexylcinnamaldehyde, techn. 85%, are performed twice a year in the laboratory.

Parameter:

SI

Remarks:

³H-thymidine incorporation Stimulation Index

Value:

2.23

Test group / Remarks:

5% preparation of the test substance in DMF

Parameter:

SI

Remarks:

³H-thymidine incorporation Stimulation Index

Value:

1.94

Test group / Remarks:

10% preparation of the test substance in DMF

Parameter:

SI

Remarks:

³H-thymidine incorporation Stimulation Index

Value:

5.11

Test group / Remarks:

25% preparation of the test substance in DMF

Parameter:

EC3

Value:

12.8

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
- 25% preparation in DMF:
-induced a biologically relevant (increase to 3-fold or above of control value = stimulation index (SI) ≥ 3)) and statistically significant increase of 3H-thymidine incorporation into the cells from the auricular lymph nodes.
-induced a biologically relevant and statistically significant response (increase to 1.5-fold or above of control value = stimulation index (SI) ≥ 1.5) in the auricular lymph node cell count
-Slight or moderate compound residues on the ears were observed in all animals of the 25% concentration during the whole observation period
- 10% preparation in DMF:
-increase of 3H-thymidine incorporation were statistically significant but failed to reach the cutoff.
- 5% preparation in DMF:
-increase of 3H-thymidine incorporation were statistically significant but failed to reach the cutoff.
-increase in the auricular lymph node cell count was statistically significant but failed to reach the cutoff
- all concentrations:
-statistically significant increases in lymph node weights
-no relevant increases (SI ≥ 1.25) in ear weights, demonstrating the absence of excessive ear skin irritation, but the increases were statistically significant at all concentrations.

DETAILS ON STIMULATION INDEX CALCULATION : The stimulation indices of 3H-thymidine incorporation, cell count, lymph node weight and ear weight measurements were calculated by dividing the mean values per test group and/or single animal values by the mean of the vehicle treated group.

EC3 CALCULATION : The EC 3 (estimated concentration that leads to the SI of 3.0) for 3H-thymidine incorporation and the EC 1.5 (estimated concentration that leads to the SI of 1.5) for cell count was calculated by linear regression from the results of all concentrations to be 12.8% and 12.1%, respectively.

CLINICAL OBSERVATIONS:
- No signs of systemic toxicity were noticed in all animals during general observation.

BODY WEIGHTS
- No relevant influence on the mean body weights

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

It is concluded that the test substance exhibits a skin sensitizing potential in the Local Lymph Node Assay under the test conditions chosen.
The threshold concentration for sensitization induction was >10% <25%. The EC 3 (estimated concentration that leads to the SI of 3.0) for 3H-thymidine incorporation and the EC 1.5 (estimated concentration that leads to the SI of 1.5) for cell count was calculated by linear regression from the results of all concentrations to be 12.8% and 12.1%, respectively.

Executive summary:

The skin sensitizing potential of the test substance was assessed using the radioactive Local Lymph Node Assay. The assay simulates the induction phase for skin sensitization in mice. It determines the response of cells in the auricular lymph nodes to repeated application of the test substance to the dorsal skin of the ears.

Groups of 5 female CBA/CaOlaHsd mice each were treated with 5%, 10% and 25% (w/w) preparations of the test substance in N,N-Dimethylformamide (DMF) or with the vehicle alone. The highest concentration was selected based on the presence of ear skin irritation in a pretest using a 50% test-substance concentration.

Each test animal was treated with 25 μL per ear of the appropriate test-substance preparation or the vehicle alone, applied to the dorsal surfaces of both ears on three consecutive days.

Three days after the last application, about 20 μCi 3H-thymidine in 250 μL sterile saline were injected into the tail vein of the mice. About 5 hours after the 3H-thymidine injection, the mice were sacrificed, and the auricular lymph nodes were removed. Lymph node response was evaluated measuring 3H-thymidine incorporation (indicator of cell proliferation). The cell count and weight of each animal’s pooled lymph nodes were also determined. In addition, a 0.8 cm diameter sample was punched out of the apical part of each ear, and for each animal, the weight of the pooled punches was determined to obtain an indication of possible skin irritation.

The mean stimulation indices (expressed as multiples of the vehicle control) for 3H-thymidine incorporation, cell count, lymph node weight and ear weight are summarized for each test group are as followed:

- 5% in DMF:

³H-thymidine incorporation Stimulation Index: 2.23

Cell Count Stimulation Index: 1.42

Lymph Node Weight Stimulation Index: 1.33

Ear Weight Stimulation Index: 1.13

- 10% in DMF:

³H-thymidine incorporation Stimulation Index: 1.94

Cell Count Stimulation Index: 1.24

Lymph Node Weight Stimulation Index: 1.17

Ear Weight Stimulation Index: 1.09

- 25% in DMF:

³H-thymidine incorporation Stimulation Index: 5.11

Cell Count Stimulation Index: 1.96

Lymph Node Weight Stimulation Index: 1.60

Ear Weight Stimulation Index: 1.09

No signs of systemic toxicity were noticed in all animals during general observation.

When applied as 25% preparation in DMF, the test substance induced a biologically relevant (increase to 3-fold or above of control value = stimulation index (SI) ≥ 3)) and statistically significant increase of 3H-thymidine incorporation into the cells from the auricular lymph nodes. The increases at the 5% and 10% test-substance preparations were statistically significant but failed to reach the cutoff.

Concomitantly, the 25% test-substance preparation induced a biologically relevant and statistically significant response (increase to 1.5-fold or above of control value = stimulation index (SI) ≥ 1.5) in the auricular lymph node cell count. The increase at 5% was statistically significant but failed to reach the cutoff.

In addition, statistically significant increases in lymph node weights were noted at all concentrations.

The test-substance concentrations did not cause relevant increases (SI ≥ 1.25) in ear weights, demonstrating the absence of excessive ear skin irritation. However, the increases were statistically significant at all concentrations. Slight or moderate compound residues on the ears were observed in all animals of the 25% concentration during the whole observation period.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. An EC3 of 12.8% was observed in the LLNA (OECD 429). Therefore, the substance is classified as a skin sensitizer of moderate potency (GHS Cat. 1B) under Regulation (EC) No. 1272/2008,as amended for the fourteenth time in Regulation (EC) No. 2020/217.